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首页> 外文期刊>Journal of Molecular Biology >DNA polymerase X from African swine fever virus: Quantitative analysis of the enzyme-ssDNA interactions and the functional structure of the complex
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DNA polymerase X from African swine fever virus: Quantitative analysis of the enzyme-ssDNA interactions and the functional structure of the complex

机译：非洲猪瘟病毒的DNA聚合酶X：酶-ssDNA相互作用和复合物功能结构的定量分析

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Interactions of polymerase X from African swine fever virus with single-stranded DNA (ssDNA) have been studied, using quantitative fluorescence titration and analytical ultracentrifugation techniques. Experiments were performed with a fluorescent etheno-derivative of ssDNA oligomers. Studies of unmodified ssDNA oligomers were carried out using the competition titration method. The total site-size of the pol X-ssDNA complex is 16(+/- 1) nucleotide residues. The large total ssDNA-binding site has a complex heterogeneous structure. It contains the proper ssDNA-binding site that encompasses only 7(+/- 1) residues. As the length of the ssDNA increases, the enzyme engages an additional binding area in interactions with the DNA, at a distance of similar to 7-8 nucleotides from the proper site, which is located asymmetrically within the polymerase molecule. As a result, the net ion release accompanying the interactions with the DNA, increases from similar to 1 for the proper DNA-binding site to similar to 6 for the total DNA-binding site. Unlike in the case of the mammalian polymerase P that belongs to the same polymerase X family, the DNA-binding areas within the total DNA-binding site of pol X are not autonomous. Consequently, the enzyme does not form different binding modes with different numbers of occluded nucleotide residues, although the interacting areas are structurally separated. The statistical thermodynamic model that accounts for the engagement of the proper and the total DNA-binding site in interactions with the DNA provides an excellent description of the binding process. Pol X binds the ssDNA without detectable cooperativity and with very modest base specificity. (c) 2005 Elsevier Ltd. All rights reserved.
机译：使用定量荧光滴定和分析超离心技术研究了非洲猪瘟病毒聚合酶X与单链DNA（ssDNA）的相互作用。用ssDNA寡聚体的荧光乙烯衍生物进行实验。使用竞争滴定法对未修饰的ssDNA低聚物进行了研究。 pol X-ssDNA复合体的总位点大小是16（+/- 1）个核苷酸残基。大的总ssDNA结合位点具有复杂的异质结构。它包含适当的ssDNA结合位点，仅包含7（+/- 1）个残基。随着ssDNA长度的增加，酶与DNA相互作用时会接合一个额外的结合区域，离适当位点的距离大约为7-8个核苷酸，该位点不对称地位于聚合酶分子内。结果，伴随着与DNA相互作用的净离子释放，从适当的DNA结合位点的相似值增加到1，到总DNA结合位点的相似值增加到6。与属于同一聚合酶X家族的哺乳动物聚合酶P的情况不同，pol X的总DNA结合位点内的DNA结合区域不是自治的。因此，尽管相互作用区域在结构上是分开的，但是该酶不会与具有不同数量的被封闭的核苷酸残基形成不同的结合模式。统计热力学模型说明了正确的和总的DNA结合位点在与DNA相互作用中的参与，为结合过程提供了出色的描述。 Pol X结合ssDNA时没有可检测的协同作用，并且碱基特异性非常适中。 （c）2005 Elsevier Ltd.保留所有权利。

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